The agricultural and pharmaceutical industry seeks production of compounds in high yield with good optical purity. The products of the present invention are useful as precursors for chemicals of high value in these industries. Specifically, cis-4-hydroxy-D-proline (CHDP) and trans-4-hydroxy-L-proline (THLP) are useful for the preparation of agrochemicals and pharmaceuticals.
CHDP is prepared commercially by chemically epimerizing THLP (Greenstein, J. P. and Winitz, M., Chemistry of the Amino Acids, vol. 3, chapter 29, John Wiley and Sons: New York (1961)). Required for the synthesis of CHDP, THLP is prepared commercially from hydrolyzed animal gelatin containing approximately 13% of the desired amino acid (U.S. Pat. No. 3,860,607). The overall process to produce CHDP involves expensive chromatographic separation of THLP from the gelatin hydrolysate and produces an excessive amount of waste relative to the amount of CHDP produced.
Alternate methods of preparing CHDP that start from relatively inexpensive starting materials and that produce fewer byproducts and waste than the THLP-based process described above are highly desirable. Several such methods have been reported. A mixture of THLP and CHDP was prepared in six steps and an overall yield of 23% from D-glutamic acid; CHDP was separated from THLP by fractional crystallization (Eguchi et al., Bull. Chem. Soc. Japan 47:1704-1708 (1986)). An enantioselective multistep synthesis of either CHDP or cis-4-hydroxy-L-proline (CHLP) from the chiral synthon (6S) and (6R) 6-methyl-4-N-((S)-1-phenylethyl)-1,4-morpholine-2,5-dione has also been reported (Madua et al., Tetrahedron: Asymmetry 7:825-830 (1996)).
Preparations of CHLP from L-aspartic acid (Burger et al., Angew. Chem. Int. Ed. Engl. 32:285-287 (1993)), from hippuric acid (-)-menthyl ester (Mehlfuhrer et al., J. Chem. Soc. Chem. Commun. 11:1291 (1994)), and from THLP have been reported (Seki et al., Biosci. Biotech. Biochem. 59:1161-1162 (1995); Papaioannou et al., Acta Chemica Scandinavica 44:243-251 (1990); Anderson et al., J. Org. Chem. 61:7955-7958 (1996)). Diastereomeric mixtures of hydroxyprolines can be prepared from epichlorohydrin (Leuchs et al., Chem. Ber. 38:1937 (1905)), and from allyl bromide (Lee et al., Bull. Chem. Soc. Japan 46:2924 (1973)), and racemic mixtures of 4-oxoprolines have been prepared from N-carboethoxyglycine ethyl ester and diethyl fumarate (Kuhn et al., Chem. Ber. 89:1423 (1956)). These and similar methods for the production of racemic mixtures of hydroxyprolines have been reviewed by Greenstein and Winitz (In, Chemistry of the Amino Acids, vol. 3, chapter 29, John Wiley and Sons: New York (1961)) and T. Kaneko (In, Synth. Prod. Util. Amino Acids, "Synthetic Methods for Individual Amino Acids. 13. Hydroxyproline", (Kaneko, T., Izumi, Y., and Chibata, I. eds.) pp. 123-127, Kodansha Ltd.: Tokyo, Japan (1974)).
The use of esterases, lipases and proteases to perform kinetic resolutions of mixtures of enantiomers or diastereomers is well known. The enantioselective hydrolysis of amino acid esters for the resolving racemic amino acids has been reported using a variety of lipases. J.-H. Houng et al., (Chirality 8:418-422 (1996)) have described the use of lipases from Rhizopus sp., Pseudomonas sp., and porcine pancreas for the kinetic resolution of N-terminal free amino acids via hydrolysis of their esters, and examined the effect of changes in the ester moiety on the enantioselectivity of the lipases. The observed enantioselectivities were highly dependent on the choice of amino acid, ester moiety and the lipase, where enantiomeric excess of remaining substrates from racemic mixtures ranged from 0 to 100%. A.-I. Chiou et al. (Biotechnology Letters 14:461-464 (1992)) have examined the enantioselective hydrolysis of hydrophobic Z-D, L-amino acid methyl esters using lipases from Aspergillus niger, Geotrichum candidum, Pseudomonas sp., and Candida cylindracea, as well as subtilisin Carlsberg type VIII protease; the observed enantioselectivities were again highly dependent on the choice of amino acid and the lipase, where enantiomeric excess of remaining substrates ranged from 3 to 92%. T. Miyazawa et al. (J. Chem. Soc. Chem. Commun. 17:1214-1215 (1988)) have described the use of lipases from Aspergillus niger, Pseudomonas fluorescens, and Candida cylindracea for the optical resolution of several unusual N-(benzyloxycarbonyl)-amino acid 2-chloroethyl esters, and noted that the enantioselectivities varied markedly with the enzymes used, and for each enzyme, on the structure of the amino acid; enantiomeric excess of the products ranged from 7 to 95%.
Many additional examples using lipases, as well as esterases and proteases, demonstrate an enantioselective resolution of a mixture of enantiomers (or diastereomers) is highly dependent on not only the choice of enzyme, but also on the chemical structure of the enzyme substrate(s). The optimal choice of enzyme and substrate is therefore not easily predicted, but requires a careful screening of a variety of enzymes while varying the chemical structure of potential substrates.